脑胶质瘤IDH1基因突变HRM检测方法的初步建立  

Preliminary Establishment of High Resolution Melting Analysis for Detecting IDH1 Gene Mutations in Gliomas

作　　者：王银辉[1,2] 张海燕[2] 杨传红[2] 张伟[3] 赖晃文[2] 陈晓东[3] 王捷[2] 

机构地区：[1]华南理工大学生物科学与工程学院,中国广东广州510006 [2]广州军区广州总医院医学实验科,中国广东广州510010 [3]广州军区广州总医院病理科,中国广东广州510010

出　　处：《生命科学研究》2013年第5期394-400,共7页Life Science Research

摘　　要：在大多数弥漫性人脑胶质瘤中常伴随着异柠檬酸脱氢酶基因(isocitrate dehydrogenase gene)1(IDH1)的突变.有着此突变的胶质瘤患者在临床上有良好的预后效果,因此可以作为胶质瘤患者的分子标记.通过提取石蜡切片中的胶质瘤组织DNA,优化条件以初步建立IDH1基因突变高分辨率熔解曲线分析法(high resolution melting,HRM)检测方法.用HRM对胶质瘤石蜡包埋组织标本中IDH1基因检测突变,并用直接测序法对比,观察反应体系和反应条件的可行性.正交试验结果证明引物最适退火温度为53℃,20μL HRM反应体系中,0.6μmol/L引物,2.5 mmol/L Mg2+,60 ng DNA模板为最佳.HRM IDH1基因突变的结果和直接测序法结果一致,但灵敏度更高.说明此HRM方法简单,特异性强,准确可靠,可为IDH1突变检测的临床应用提供借鉴依据.Mutations in the isocitrate dehydrogenase 1 （IDH1） genes have been identified in a large propor- tion of diffuse gliomas. Since the mutations have been associated with better clinical outcome, they are suitable predictive markers for gliomas patients. It was performed with optimizing conditions to preliminary establish a high resolution melting for the detecting of IDH1 mutations in routine formalin-fixed tissues of brain biop- sies. IDH1 mutations in paraffin-embedded tissues of gliomas were detected by HRM and direct sequencing, estimating the feasibility of reaction system and reaction conditions. The optimal annealing temperature was 53 ℃. The optimization of 20 μL-HRM reaction system including 0.6 μmol/L primer, 2.5 mmol/L Mg2＋, 60 ng DNA template-, was established by Orthogonal Design. The results were consistent with the direct se- quencing but have a higher sensitivity. It showed that HRM was simple, specific, accurate and reliable method. It would provide the reference for the clinical application of detection IDHI mutations.

关 键 词：异柠檬酸脱氢酶基因突变 高分辨率熔解曲线 胶质瘤检测 

分 类 号：Q331[生物学—遗传学]